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PCK1 Negatively Regulates Cell Cycle Progression and Hepatoma Cell

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PCK1 Negatively Regulates Cell Cycle Progression and Hepatoma Cell Tuo et al. Journal of Experimental & Clinical Cancer Research (2019) 38:50 https://doi.org/10.1186/s13046-019-1029-y RESEARCH Open Access PCK1 negatively regulates cell cycle progression and hepatoma cell proliferation via the AMPK/p27Kip1 axis Lin Tuo†, Jin Xiang†, Xuanming Pan†, Jieli Hu, Hua Tang, Li Liang, Jie Xia, Yuan Hu, Wenlu Zhang, Ailong Huang*, Kai Wang* and Ni Tang* Abstract Background: Altered glucose metabolism endows tumor cells with metabolic flexibility for biosynthesis requirements. Phosphoenolpyruvate carboxykinase 1 (PCK1), a key enzyme in the gluconeogenesis pathway, is downregulated in hepatocellular carcinoma (HCC) and predicts poor prognosis. Overexpression of PCK1 has been shown to suppress liver tumor growth, but the underlying mechanism remains unclear. Methods: mRNA and protein expression patterns of PCK1, AMPK, pAMPK, and the CDK/Rb/E2F pathway were determined using qRT-PCR and western blotting. Cell proliferation ability and cell cycle were assessed by MTS assay and flow cytometric analysis. The effect of PCK1 on tumor growth was examined in xenograft implantation models. Results: Both gain and loss-of-function experiments demonstrated that PCK1 deficiency promotes hepatoma cell proliferation through inactivation of AMPK, suppression of p27Kip1 expression, and stimulation of the CDK/Rb/E2F pathway, thereby accelerating cell cycle transitionfromtheG1toSphaseunderglucose-starved conditions. Overexpression of PCK1 reduced cellular ATP levels and enhanced AMPK phosphorylation and p27Kip1 expression but decreased Rb phosphorylation, leading to cell cycle arrest at G1. AMPK knockdown significantly reversed G1-phase arrest and growth inhibition of PCK1-expressing SK-Hep1 cells. In addition, the AMPK activator metformin remarkably suppressed the growth of PCK1-knockout PLC/PRF/5 cells and inhibited tumor growth in an orthotropic HCC mouse model. Conclusion: This study revealed that PCK1 negatively regulates cell cycle progression and hepatoma cell proliferation via the AMPK/p27Kip1 axis and supports a potential therapeutic and protective effect of metformin on HCC. Keywords: Phosphoenolpyruvate carboxykinase 1, Hepatocellular carcinoma, Cell cycle arrest, CDK/Rb/E2F pathway, AMP-activated protein kinase Background of oxaloacetate (OAA) and GTP to phosphoenolpyruvate Metabolic reprogramming or “deregulating cellular ener- (PEP) and GDP by phosphoenolpyruvate carboxykinase getics” is a hallmark of cancer [1]. Tumor cells undergo (PCK; also known as PEPCK, EC number 4.1.1.32) is the aerobic glycolysis even in the presence of oxygen, also first rate-limiting step in hepatic gluconeogenesis [3, 4]. known as the Warburg effect, which is critical for meet- Two isoforms of PCK have been described, a cytoplas- ing the metabolic requirements of rapid cancer cell mic form (PCK1, PEPCK-C) and a mitochondrial iso- growth and proliferation [2]. The liver is the major site form (PCK2, PEPCK-M) [5]. In the mammalian liver, of gluconeogenesis during fasting, where the conversion PCK1 accounts for over 95% of gluconeogenesis activity [6], is upregulated in colon and melanoma cancers, and * Correspondence: [email protected]; [email protected]; is associated with increased glucose consumption to [email protected] † support anabolic metabolism and tumor growth. How- Lin Tuo, Jin Xiang and Xuanming Pan contributed equally to this work. ever, in hepatocellular carcinoma (HCC), the expression Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, levels of PCK and other key gluconeogenic enzymes are The Second Affiliated Hospital, Chongqing Medical University, Chongqing, strikingly suppressed [7, 8], indicating that PCK has People’s Republic of China © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tuo et al. Journal of Experimental & Clinical Cancer Research (2019) 38:50 Page 2 of 16 contrasting roles in tumorigenesis in different organs. Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) Nevertheless, the role of PCK in HCC progression re- supplemented with 10% fetal bovine serum (FBS; Gibco, mains incompletely understood. Rockville, MD, USA), 100 IU penicillin, and 100 mg/mL Recent metabolomic studies confirmed that glucose levels streptomycin. HepG2 cells were cultured in minimum in tumors are generally lower than those in non-transformed essential medium (MEM; Hyclone). Cell lines involved tissues [9, 10]. However, cancer cells require sufficient glu- in this study were recently authenticated by short tan- cose to facilitate rapid tumor growth through the generation dem repeat (STR) profiling (Beijing Microread Gene of building blocks, which are necessary for the synthesis of Technology Co., Beijing, China). All cells were incubated essential cellular components [9, 11]. Therefore, cancer cells in a humidified atmosphere at 37 °C containing 5% CO2. require delicate energy sensing and metabolic adaptation For cell cycle analysis, cells were collected at different pathways. Mammalian AMP-activated protein kinase time points post-synchronization. For low-glucose treat- (AMPK) is a central cellular sensor of adenine nucleotides ment, cells were incubated in glucose-free DMEM that plays a key role in sensing cellular energy availability (Hyclone) supplemented with 10% FBS, 100 IU penicillin, and regulating metabolic adaptation pathways [11, 12]. En- 100 mg/mL streptomycin, and the indicated concentra- ergy stress conditions in tumor tissues are sensed by AMPK tions of glucose (Hyclone) for 12 h. For immunoblotting through elevated intracellularAMP/ATPratios.Onceacti- and cell cycle analysis under glucose-deprived conditions, vated, phosphorylated AMPK acts to restore the energy bal- cells were incubated in DMEM containing 1 mM glucose ance by increasing catabolic ATP-generating processes, such and metformin (5 mM; Cat# S1950; Selleck, Houston, TX, as glycolysis and fatty-acid oxidation, while decreasing USA) for 12 h and then collected for analysis. For prolifer- ATP-consuming processes, such as fatty acid synthesis and ation and colony formation assays, cells were cultured in cell proliferation [13]. AMPK activation has been reported to normal DMEM supplemented with 0.5 mM metformin. suppress cell proliferation by regulating cell cycle progression or inhibiting the synthesis of certain proteins, such as Plasmid constructs and adenovirus production p27Kip1 [14–16]. The full-length cDNA of PCK1 (coding sequence of In this study, we explored the potential role of PCK1 in NM_002591) was amplified from plasmid pOTB7-PCK1 the suppression of cellular proliferation and tumor growth (Cat# FL07339; GeneCopoeia, Rockville, MD, USA). in HCC. Interestingly, we found that PCK1 activates Primers are listed in Additional file 1: Table S1. The amp- AMPK and delays G1 to S phase (G1/S) transition. Our lified PCK1 fragment was inserted into the shuttle vector study also revealed that the AMPK activator metformin pAdTrack-TO4 (kindly provided by Dr. Tong-Chuan He, remarkably suppresses tumor growth in an orthotropic University of Chicago, USA). Adenoviral recombinant HCC mouse model. pAd-PCK1 was generated using the AdEasy system as described previously [17]. The analogous adenovirus Methods expressing GFP only (AdGFP) was used as control. Patient samples Liver tumor specimens and adjacent non-tumorous Lentivirus construction specimens were collected from 20 patients who under- Two pairs of oligonucleotides encoding short hairpin went surgical resection at the Second Affiliated Hospital RNA (shRNA) targeting isoform α1 of AMPK (Additional of Chongqing Medical University (Chongqing, China). file 1: Table S1) were designed and subcloned into the len- Patients had not received chemotherapy or radiation tiviral vector pLL3.7 (kindly gifted by Prof. Bing Sun from therapy before surgery. The samples were frozen imme- the Shanghai Institute of Biochemistry and Cell Biology, diately after surgery and stored in liquid nitrogen. All Chinese Academy of Sciences, China) to generate patients provided informed consent and the study was shAMPK lentivirus. A control virus (shControl), with a conducted with the approval of the Institutional Ethical scrambled shRNA insert, was also generated. For lentiviral Review Board of Chongqing Medical University (project supernatant production, HEK-293 T cells were transfected license number: 2017012). with 2 μgVSVG,3μg Δ8.9, and 4 μg of the shRNA lenti- viral construct for lentiviral package using Lipofectamine Cell cultures and drug treatment 2000 reagent (Thermo Fisher Scientific, Waltham, MA, The human hepatoma cell lines HepG2, SK-Hep1, and USA), according to manufacturer’s instructions. SK-Hep1 PLC/PRF/5 were obtained from the American Type Cul- cells were transduced with the packaged lentiviruses in ture Collection (ATCC; Manassas, VA, USA). Other the presence of 5 μg/mL polybrene. hepatoma cell lines like Huh7 and normal human liver cells MiHA were obtained from the
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